Genetic differentiation and phylogeography of Erythroneurini (Hemiptera, Cicadellidae, Typhlocybinae) in the southwestern karst area of China

Abstract Erythroneurini is the largest tribe of the microleafhopper subfamily Typhlocybinae. Most prior research on this tribe has focused on traditional classification, phylogeny, and control of agricultural pests, and the phylogeography of the group remains poorly understood. In this study, the mitochondrial genomes of 10 erythroneurine species were sequenced, and sequences of four genes were obtained for 12 geographical populations of Seriana bacilla. The new sequence data were combined with previously available mitochondrial DNA sequence data and analyzed using Bayesian and Maximum‐Likelihood‐based phylogenetic methods to elucidate relationships among genera and species and estimate divergence times. Seriana was shown to be derived from within Empoascanara. Phylogeographic and population genetic analysis of the endemic Chinese species Seriana bacilla suggest that the species diverged about 54.85 Mya (95% HPD: 20.76–66.23 million years) in the Paleogene period and that population divergence occurred within the last 14 million years. Ancestral area reconstruction indicates that Seriana bacilla may have originated in the central region of Guizhou, and geographical barriers are the main factors affecting gene flow among populations. Ecological niche modeling using the MaxEnt model suggests that the distribution of the species was more restricted in the past but is likely to expand in the future years 2050 and 2070.


| INTRODUC TI ON
Leafhoppers (Cicadellidae) are one of the largest families of plant-feeding insects.They are generally between 3 and 15 mm in length, small in size, widely distributed, and occur in most habitats where vascular plants are found.Many leafhopper species transmit plant-pathogenic bacteria and viruses that affect various crops, grasslands, forest trees, fruit trees, and other economic plants (Roddee et al., 2018).Adult and juvenile (nymph) leafhoppers usually have similar ecology, living on the aboveground parts of their host plants.Nymphs are capable of jumping but generally do not disperse over long distances.Adults usually have well-developed wings but relatively few species regularly migrate over long distances.Leafhopper distribution is often constrained by barriers to dispersal such as mountains, water bodies, or regions of unsuitable habitat.Previous studies of leafhopper biogeography (e.g., Cao et al., 2023;Krishnankutty et al., 2016;Wang et al., 2017) have revealed considerable largescale biogeographic structure, with many diverse lineages restricted to particular biogeographic regions.Few studies have examined smaller-scale biogeographic patterns among species within a single genus (e.g., Bernt et al., 2013;Zhang et al., 2016) and studies of phylogeographic patterns within individual leafhopper species have, so far, focused only on agricultural pests (e.g., Akmal et al., 2018;Zhang et al., 2020).Due to their limited dispersal ability, abundance, and specialization on certain host plants and habitats, leafhoppers are ideal subjects for research on speciation and phylogeography (Hill et al., 2009;Marshall et al., 2008).Most research on leafhoppers continues to focus on morphology-based taxonomy and control of disease vectors and other agricultural pests (Dietrich, 2013).However, additional research is needed to elucidate the particular evolutionary mechanisms that gave rise to the great diversity of leafhopper species.Phylogeography can help improve understanding of the contributions of dispersal and geographic barriers to genetic diversification and speciation in leafhoppers.
Phylogeography combines biogeographic knowledge and molecular biology techniques to reconstruct the historical evolution and formation of different geographic populations among closely related species and within species (Avise et al., 1987;Kumar et al., 2016).
Phylogeography has made great contributions to the study of human evolution (Beaumont, 2004), species formation (Kohn, 2005), and biological conservation (O'Brien, 1994).The most widely used molecular markers in phylogeography are in the mitochondrial genome (Clarke et al., 2015;Diedericks & Daniels, 2014;Liu et al., 2016).With the development and improvement of DNA sequencing technology and evolutionary theory, phylogeography has developed rapidly and become an important tool in evolutionary biology (Beheregaray, 2008).
In view of the limited research on the population differentiation and phylogeography of leafhoppers, this study focused on Erythroneurini, which have been well studied from a taxonomic perspective but have not previously included in phylogeographic studies.Based on our extensive field surveys and sampling in the southwest karst regions of China, we conducted phylogenetic analyses to elucidate the relationships among genera and species of this tribe, reconstruct ancestral areas, and estimate divergence times.We then used finer-scale analyses to further explore the phylogeography of one species apparently endemic to this region.Seriana bacilla is a shared species in the southwest and can be used to explore genealogical geography, geological events and climatic fluctuations, population genetic differentiation, historical dynamics, and suitability zones.

| Specimen collection and DNA extraction
The Specimens used in this study were collected from 25 counties and cities in Guizhou, Yunnan, Sichuan, Guangxi, and Chongqing, situated in the karst environment of southwestern China, by sweeping and light trap (Figure 1).The collected specimens were preserved in 98% ethanol, transported to the laboratory, and stored

| Mitochondrial genome annotation and analysis
Raw sequence data were assembled using Geneous Prime V 2021.2.2 software and the mitochondrial genome loop splicing was performed manually using Geneous Prime V 2021.2.2 software after determining that the data were correct (Kearse et al., 2012).Genome assembly was performed using GetOrganelle 1.7.5 (parameters: -R 10 -k 21,45,65,85,105 -F animal_mt) (Jin et al., 2020), and the assembly results were blastn (version: BLAST 2.2.30+; parameters: −evalue 1e−5) with the proximal reference mitogenome (accession number NC_047465), and the candidate sequence assembly results were determined based on the comparison.The secondary structure of mitochondrial tRNA genes and 22 tRNA gene positions were determined using the MITOS Web server (Bernt et al., 2013) and the tRNAscan-SE (Lowe & Eddy, 1997) online website.Contigs were used as input for BLAST searches in GenBank to verify their identity as leafhopper mtDNA sequences (Altschul et al., 1997;Meng et al., 2013;Yu et al., 2017).

| Phylogenetic analysis
Two datasets were analyzed.The first, used to estimate relationships among the currently recognized tribes of Typhlocybinae, included sequences of the 13 mitochondrial protein-coding genes (PCGs).The second included sequences of four mitochondrial genes (COI, COII, Cytb, 16S rRNA) and was used to assess phylogenetic relationships between species of the genera Seriana and Empoascanara.The latter dataset was also used for population-level phylogeographic analysis of S. bacilla.
The newly obtained gene sequences were aligned and analyzed, and combined with previously available sequence data using PhyloSuite v1.2.2 (Zhang et al., 2020).Sequence alignment was performed using MAFFT v7.4 (Katoh & Standley, 2013).PartitionFinder v2.1.1 (Lanfear et al., 2017) was used to select the best-fit partitioning scheme for linked nucleotides and the corresponding nucleotide substitution model.Phylogenetic analysis was conducted using the Maximum Likelihood (ML) and Bayesian Inference (BI) methods, as implemented in IQTREE 1.6.5 (Nguyen et al., 2014) and MRBAYES 3.2 (Ronquist et al., 2012), respectively.ML analysis was run using IQ-TREE for 10,000,000 generations, checking the confidence values of each branch node of the system tree every 1000 times, and BI analysis was run independently using four Markov chain Monte Carlo (MCMC) chains (three heated chains and one cold chain) starting from a random tree; each chain was run for 2 × 10 7 generations and sampled once every 1000 generations.Tracer v1.7.1 (Rambaut et al., 2018) was used to check for effective sample size (ESS) > 200.
Visualization and further processing of phylogenetic trees were performed using FigTree 1.4.3.

| Genetic diversity analysis
The genetic diversity of S. bacilla was calculated from mitochondrial DNA sequence data by DNAsp5.0 software.In the calculation process, populations were grouped according to collection regions, and since Guangxi and Yunnan were each represented by a single sample, these two collection regions were not counted in the calculation of nucleotide diversity and genetic structure.

| Divergence time estimation
Since leafhopper fossils have not yet been included in morphological phylogenetic analysis, we use available fossils to calibrate the root nodes of their respective tribes in order to conservatively estimate the minimum ages of these groups.We used BEAST v2.6.6 (Bouckaert et al., 2014) to estimate the date of origin of Erythroneurini and the divergence time of each species using tandem mitochondrial data under the relaxed log-normal assumption of the clock and the priori Yule model.Based on the previous molecular timetree of Membracoidea (Christopher et al., 2017) and information on the oldest Membracoidea included in the Paleobiology Database (PDBD) Navigator website (http:// paleo biodb.org/ navig ator/ ), the maximum age of the root node was constrained with a relaxed lower bound of 174.1 million years ago (Mya) (Yan et al., 2022).Oligo-Miocene Dominican amber was used to provide minimum age calibration points for Evacanthinae (Dietrich & Vega, 1995).Dominican amber was also used to provide minimum age calibration points for Cicadellinae (Dietrich & Vega, 1995).Therefore, we set four fossil calibration points, (A) root age < 174.1 Mya; (B) 17.5-120 Mya; (C) 17.5-110 Mya; (D) 17.5-90 Mya (normally distributed calibration density; mean = 1 Ma).The Jmodeltest software was used to select the most suitable model, the Substitution Model was selected as the GTR model, the Tree Prior was selected as the Yule process, and the MCMC method was run for 100,000,000 generations, with sampling every 1000 generations (Drummond et al., 2012;Drummond & Rambaut, 2007).Stationarity was assessed using TRACER 1.7.1, with ESS values of >200 taken as evidence of convergence.Maximum clade credibility (MCC) trees were generated using TreeAnnotator v2.4.1 after discarding the first 10% as burn-in.Figtree software was used to export and visualize the time tree.

| Ancestral area reconstruction
To assess the origin and possible dispersal pathways of S. bacilla species, a time-calibrated phylogenetic tree obtained from the BEAST analysis with all outgroups removed and only S. bacilla species retained was used as the input tree for ancestral area analysis.BioGeoBEARS (Matzke, 2013) as implemented in RASP v3.2 (Yu et al., 2015) was used to reconstruct ancestral areas and infer their biogeographic history.Specimens representing different S. bacilla populations were assigned to eight geographic areas:  (Jiang et al., 2016;Mendlik & Gobiet, 2016;Wang & Chen, 2014).
The data obtained were processed by ArcGIS10.7 software, and the TA B L E 1 List of newly sequenced species.MaxEnt model was used to predict suitable areas.Areas are categorized into four different types: non-suitable area (0-0.07),low suitable area (0.07-0.3), medium suitable area (0.3-0.6), and high suitable area (0.6-1).
To avoid over-parameterization caused by redundant variables in the model, Pearson correlation analysis was performed on bioclimatic factors using SPSS software.When |r| > 0.9, the variable with less biological significance was eliminated.In the end, nine biocli-

| Phylogenetic analysis
Forty-seven species representing six tribes of Typhlocybinae were used as the ingroup, and four species representing two other subfamilies were used as outgroups.The species included are listed in Table 4.The ML phylogenetic tree and the BI phylogenetic tree constructed separately based on 13 PCGs showed similar topologies (Figure 2).Phylogenetic trees shows the relationship between these tribes as ((Typhlocybini + Zyginelli ni) + (Erythroneurini + Dikraneurini)) + (Empoascini + Alebrini ). Erythroneurini + Dikraneurini are monophyletic sister groups to each other and Empoascini + Alebrini are also monophyletic.
In agreement with previous studies, our phylogeny placed Seriana and Empoascanara as sister groups.To further explore their relationships, we downloaded all available mtDNA sequences for species of these genera from Genbank, and used four species from

| Genetic diversity analysis
The results of analysis based on four genes (COI, COII, Cytb, and 16S rRNA) are shown in

| Divergence-time estimation
The

| Ancestral area reconstruction
The results of ancestral area reconstruction based on Biogeobears analysis in RASP are shown in Figure 7. Testing and comparison of six models using the AICc weighting method showed that the DIVALIKE

| Species distribution model
Figure 8 shows the predicted distributions of S. bacilla during different periods.The AUC of the species distribution model is  winter provides good climatic conditions for the survival of S. bacilla.
The vegetation in the suitable areas is mostly evergreen broad-leaved forests, and the other rich vegetation types such as evergreen deciduous broad-leaved mixed forests and shrubs are also important factors affecting the range of their suitable habitat.

| DISCUSS ION
In this study, we newly obtained mitochondrial genomes for 10 erythroneurine species and sequenced four genes for representatives of 12 geographical populations of S. bacilla.The phylogenetic tree of Typhlocybinae includes all species of all genera of the six tribes recognized in Typhlocybinae for which mitochondrial DNA sequences are available on GenBank.The monophyly of Typhlocybini, Dikraneurini, Empoascini, Erythroneurini, and Alebrini is supported in our results.Song and Li (2014) reported that Seriana and Empoascanara are very similar in external appearance, with the fuscous body color, the anterior margin of crown produced medially, and usually with an irregular dark spot at anterior margin of the crown medially.Our analysis suggests that Seriana is derived from within Empoascanara but this result needs to be confirmed by analyses that include more than one species of Seriana.
Haplotype diversity was high in all populations of S. bacilla except the Chongqing population, with Guizhou having the highest nucleotide diversity, Sichuan the second highest, and Chongqing the lowest.The low genetic diversity from Chongqing may be due to a young population that has not accumulated much genetic variation, or the population may have experienced a "selective sweep" or 'founder effect' .Generally, older populations have higher genetic diversity and colonizing or invasive populations have lower levels of genetic diversity (Lanzavecchia et al., 2008;Savolainen et al., 2002), and the highest genetic diversity in Guizhou suggests that this population may be the ancestral population of S. bacilla in southwest of China, although this may also be an artifact of the larger number of samples from this region.
In the phylogenetic analysis, the Guizhou population is relatively divergent from the Yunnan and Guangxi populations, while the di- | 13 of 19   LUO et al.   to that between Yunnan and Guangxi.The genetic differentiation coefficient between the two populations in Sichuan and Guizhou is very small (F st < 0), and the gene flow is very large (N m > 4), indicating that there is no genetic differentiation due to the frequent gene exchange between the two populations.Geographical barriers between Sichuan and Guizhou are small.Based on our results, geographical barriers (or lack thereof) appear to be the main mediators of gene flow among populations of S. bacilli in China (Pyron & Burbrink, 2010;Smith et al., 2014;Ye et al., 2013).
On our molecular time tree of Typhlocybinae, the divergence time estimate for Erythroneurini (85.521 million years) is earlier than that of Yan et al. (2022) and later than that of Christopher et al. (2017).The split time between Erythroneurini and Dikraneurini in the study of Yan et al. (2022) is estimated to be 76 million years, while Christopher et al. (2017) reported 95 million years.One possible explanation for these differences is the larger numbers of taxa included in our analysis compared to the previous analyses.
The first fossil Typhlocybinae inclusions from Eocene Rovno amber, and recent molecular time trees place the origin of Cicadellidae in the Cretaceous (Christopher et al., 2023;Johnson, Dietrich, Friedrich, Beutel, et al., 2018;Johnson, Dietrich, Friedrich, Beutelet, et al., 2018;Lu et al., 2021), but most modern genera arising during the Paleogene and multiple transcontinental dispersal events occur in the Paleogene (Cao et al., 2023).The divergence of S. bacilla in the mountains, and was less affected by glaciers.The climate was mild, providing a favorable living environment for animals and plants during the glacial period (Chen et al., 2011).The Yunnan-Guizhou Plateau was not covered by glaciers during the Quaternary glacial period, and the impact of the glacial period on the region was relatively small, mainly in terms of temperature and precipitation, while a small range of environmental changes made the species migrate (Hewitt, 2000).It is speculated that the Yunnan-Guizhou Plateau and the Sichuan Basin are the refuge during the ice age.Around 0.07-0.1 million years, China was in the interglacial period when temperatures began to rise and species migrated from refugia to various locations (Xu et al., 2012).black brown, with anterior angle near scutellum orange-yellow (Figure 9).
Subgenital plate broadened at sub-base and with four long macrosetae on lateral surface (Figure 10b).Connective Y-shaped, with stem strong (Figure 10c).Style apex truncate, preapical lobe distinct (Figure 10d).Aedeagus with one pair of large processes basally; aedeagal shaft apex bifurcated; preatrium also with one pair of long processes arising from base.Gonopore located at middle part of the aedeagus shaft, ventrad (Figures 9f, 10e).

CO N FLI C T O F I NTER E S T S TATEM ENT
This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal.
We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these.
There are no conflicts of interest to declare.

1
Sample collection and distribution map (the left image shows the distribution of all spotted Erythroneurine specimen collections, and the right image shows the distribution of Seriana bacilla specimen collections).| 3 of 19 LUO et al. in a freezer at −20°C.Genomic DNA was extracted from the legs and thorax of each leafhopper specimen using the DNeasy tissue kit following manufacturer's instructions.The extracted DNA was numbered and stored at −20°C after passing the DNA quality test.Whole mitochondrial genome sequences were determined at Berry Genomics (Beijing, China) using an Illumina Novaseq 6000 platform (Illumina, Alameda, CA, USA) by 150 bp paired-end reads and 5.87 G of raw data were obtained.All specimens are stored in the collection of the School of Karst Science, Guizhou Normal University, China (GZNU).

(
A) central Yunnan; (B) northeastern Guangxi; (C) southeastern Sichuan; (D) central Sichuan; (E) western Chongqing; (F) northern Guizhou; (G) northeastern Guizhou; (H) central Guizhou.2.7 | Species distribution model Ecological niche modeling based on the current distribution of S. bacilla, we use past, present, and future bioclimatic factors to predict potential distribution areas during the Last Glacial Maximum (LGM, ~22,000 years ago), Mid-Holocene (~6000 years ago), present and the future years 2050, 2070.S. bacilla was first described by our research team in 2020 and its distribution data have mainly been collected from our team through fieldwork over several years.Distribution data of S. bacilla were also compiled from relevant theses, dissertations, and other literature, and from China Knowledge Base, 3I Interactive Key and Taxonomic Databases, and Web of Science.ArcGIS 10.7 was used to filter distribution data, and the final dataset contains data from 55 locations.Nineteen bioclimatic variables of paleoclimate are available from the World Climate Dataset v1.4 (http:// www.world clim.org) with resolution of 10 min (Hijmans et al., 2005).Considering the uncertainty of a single climate model, the bioclimatic factors for 2050 and 2070 in the paper use two representative concentration pathways (RCPs) from the CCSM4 model and MIROC-ESM model, that is, RCP26 and RCP45 13PCGs dataset comprising 47 species of Typhlocybinae was used to estimate divergence times.As shown in the cladogram (Figure 5), the divergence time of Typhlocybinae is 113.59 million years in the middle Cretaceous, and the 95% confidence interval (95% high posterior density, i.e., 95% HPD) is 111.61-115.48 million years; the internal clades begin to diversify at 100.34 million years (95% HPD: 63.98-108.82 million years), the earliest being the Empoascini and Alebrini at 90.77 million years (95% HPD: 64.09-103.01 million years); the Typhlocybini and Zyginellini at 79.26 million years (95% HPD: 36.9-90.21 million years); the Erythroneurini and Dikraneurini are 85.521 million years (95% HPD: 43.54-97.34 million years); and the divergence time of species between the genera of Seriana and Empoascanara is about 54.85 million years (95% HPD: 20.76-66.23 million years).Divergence times were estimated for the different geographic populations of S. bacilla, and the main divergence time is shown in Figure 6.The divergence time for S. bacilla was about 54.832 Mya (95% HPD: 52.866-56.762 million years) during the Paleoproterozoic period, and this result differs somewhat from the results in Figure 5, but is within the 95% confidence interval.The branches began to evolve about 14.317 Mya (95% HPD: 3.144-30.95 million years) in the Neoproterozoic, with evolutionary clade 1 diverging first and then internally into two branches at about 9.278 million years (95% HPD: 1.723-22.174 million years); evolutionary clade 2 began to differentiate internally in 6.624(95% HPD: 1.021-18.636 million years).
Phylogenetic tree from Typhlocybinae based on nucleotide sequence of 13 PCGs.The first and second numbers to the left of each node denote the ultrafast bootstrap value (UBP) for ML analysis and the Bayesian posterior probability (BPP) for Bayesian inference.F I G U R E 3 Phylogenetic tree of S. bacilla and closely related species constructed based on four mitochondrial genes (COI, COII, Cytb, 16S rRNA).The first and second numbers to the left of each node denote the ultrafast bootstrap value (UBP) for ML analysis and the Bayesian posterior probability (BPP) for Bayesian inference.F I G U R E 4 Phylogenetic tree of 12 geographical populations of S. bacilla constructed based on combined gene sequences (COI, COII, Cytb, 16S rRNA).The first and second numbers to the left of each node denote the ultrafast bootstrap value (UBP) for ML analysis and the Bayesian posterior probability (BPP) for Bayesian inference.
vergence between Yunnan and Guangxi populations is relatively low.The Yunnan-Guizhou Plateau is a natural geographical barrier between the three populations.The terrain of the Yunnan-Guizhou Plateau, which is high in the northwest and low in the southeast, may hinder gene flow between Yunnan and Guizhou compared F I G U R E 6 Divergence time tree (Mya) of different geographical populations of S. bacilla constructed based on COI, COII, Cytb, and 16S rRNA.
southwest was also shown to have occurred about 54.832 Mya during the Paleogene.The divergence of individual populations of this species occurred between 3 and 14 Mya.The divergence within individual branches mostly occurred in the range of 0.329-4.601 million years, with most occurring during the Pleistocene period and a few during the Pliocene.This means that the divergence of the species was mainly influenced by the Quaternary ice age.The climatic upheavals caused by the repeated alternation of Quaternary ice ages had a great impact on the evolution as well as the distribution of flora and fauna in China (Zhang et al., 2016).The uplift of the Tibetan Plateau caused by the collisional compression of the Indian and Eurasian continental plates has greatly altered the environment and climate of southwest China (Favre et al., 2015).During the Pliocene 3-4 million years, the rapid uplift of the Hengduan Mountain Range, the East Asian monsoon was once again strengthened, temperatures dropped, and species migrated to warmer regions.In the Eocene, about 30 Mya, the Sichuan Basin formed due to the uplift of the Qinghai-Tibet Plateau was separated by Qinling Mountain, Daba Mountain, Yunnan-Guizhou Plateau and other F I G U R E 7 Results of Ancestral area reconstruction of S. bacilla based on RASP.F I G U R E 8 Potential distribution areas of S. bacilla in different geological periods using MaxEnt model with bioclimatic factors.(a) Last glacial maximum (LGM); (b), Mid-Holocene; (c), Present day; (d), year 2050; (e), year 2070.

4. 1
| Taxonomy 4.1.1| Empoascanara distant, 1918 Empoascanara Distant, 1918: 94.Type species.Empoascanara prima Distant, 1918.Crown is slightly wider, equal in width, or slightly narrower than the widest part of pronotum.Vertex is blunted and slightly produced medially, about half as long as pronotum, often with variable black spots in the central part.Pronotum is often brownish yellow, with orange red tint, or dark only along posterior margin.Male genitalia: Pygofer dorsal appendages with diverse shapes, articulately movable or immovably fused to dorsal margin.Pygofer lobe is smooth or angulated.Subgenital plate expanded basally and characterized by 2-4 macrosetae along outer margin, and bears distinct rigid setae along upper margin form a continuous row from subbase to apex.
List of mitochondrial genomes used for phylogenetic analysis of Typhrocybinae in this study.
Typhlocybini as outgroups for a total of 19 species to construct ML and BI phylogenetic trees based on four genes (COI, COII, Cytb, 16S rRNA).The topologies from ML and BI analyses are consistent as follows: E. gracilis is sister to the remaining species; E. sipra, E. wengangensis, and E. circumscripta are clustered together; E. defecta and E. bidenticulatas sp.n. are sister groups, E. dissimilis and E. plamka are sister groups; E. alami, E. hongkongica, and E. dwalata are in one clade; E. falcata and E. quarta are sister groups, and E. angkhangica and S.rRNA.According to the clustering of S. bacilla in the phylogenetic tree (Figure4), it was divided into two major branches.Clade 2 was dominated by S. bac CQYC, S. bac CQJLP, S. bac SCNJ, S. bac GZZY, S. bac GZTR, and S. bac SCYA, S. bac YNYX and S. bac GXGL, but support for this branch was low.S. bac SCLS, S. bac GZSB, S. bac GZGY, and

Table 5
. All populations had high haplotype diversity (1.000), except for Chongqing, where it was low; nucleotide diversity was highest in Guizhou (0.014, 0.024, 0018, 0.008, 0.15), followed by Sichuan (0.01, 0.016, 0.009, 0.008, 0.01), and lowest in Chongqing.The genetic differentiation coefficient (F st ) and gene flow (N m ) of S. bacilla were calculated based on different genes using Arlequin software, and the results are shown in Table6.In the results of COI, 16S rRNA genes and the combined gene in Guizhou was the best-fitting model.The results indicate that the most recent common ancestor of the sampled populations of S.
bacilla was most likely distributed in the central region of Guizhou.From there, this species appears to have spread to central Sichuan, then into northeast Guangxi and central Yunnan, to northeast Guizhou.The species then spread to western Chongqing, and finally to northern Guizhou.
Genetic diversity analysis of different geographical populations of S. bacilla calculated based on mitochondrial genes.Genetic differentiation coefficient (F st ) and gene flow (N m ) between collection regions calculated based on different genes.Below the diagonal: F st values, above the diagonal: N m values.
>0.9, indicating that the model has good performance.In LGM, the potential suitable area of S. bacilla is 116.250 × 10 4 km 2 , accounting for 12.109% of the total terrestrial area of China, of the total area of the suitable areas showed an increasing trend, reaching 191.105 × 10 4 km 2 , accounting for 19.907% of the total land area of China.In the future, suitable areas for S. bacilla in China are predicted to increase.It is worth noting that the suitable habitat for S. bacilla is mostly in slightly humid areas.The subtropical monsoon climate with high temperature and rainfall in summer and mild and low rainfall in TA B L E 5 TA B L E 6 The suitable habitat area of S. bacilla at different periods predicted based on MaxEnt model.
4.1.2| Empoascanara bidenticulata Luo & Song, sp.nov.Description: Description: Vertex brownish yellow, with a single large black patch in the middle of anterior margin.Eyes black (Figure 9a).Pronotum orange yellow, with dark posterior margin.Scutellum orangeyellow, with basal triangles black.Forewing TA B L E 7 Abbreviations: LGM, Last glacial maximum in the past; MH, Mid-holocene.